An optimized protocol for stepwise optimization of real-time RT-PCR analysis

摘要

The PCR efficiency (E; %) for each primer pair was calculated as E = (10-1/A − 1) × 100. The cDNA concentration in 1:10, 1:20, 1:40, 1:80, and 1:160 dilutions was 5, 2.5, 1.25, 0.625, 0.3125 ng/µl, respectively, while the Log (cDNA in ng/reaction) for the 1:10, 1:20, 1:40, 1:80, and 1:160 dilutions were 0.69897, 0.39794, 0.09691, −0.20412, and −0.50515, respectively. The data from the lowest (or highest) one (or two) cDNA concentration might have been omitted in order to obtain R2 ≥ 0.99 and E = 100 ± 5% for the data from the remaining four (or three) consecutive cDNA concentrations for the best primer pair for each candidate gene. This served as the prerequisite for using the 2−ΔΔCt method for data analysis